uHTS identification of inhibitors of cullin neddylation in a TR-FRET assay
Cullin-RING ubiquitin ligases (CRLs) are responsible for ~20% of all intracellular protein degradation by the proteasome. This critical role in maintaining protein homeostasis is emphasized by links between CRLs and diseases such as cancer and neurodegenerative disorders, inflammation, host-pathogen interactions, and the immune response. The activation of CRLs by covalent modification with the more ..
BioActive Compounds: 2123
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute(SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: R03 DA034599-01
Assay Provider: Matthew D. Petroski, Ph.D., Sanford-Burnham Medical Research Institute
Cullin-RING ubiquitin ligases (CRLs) are responsible for ~20% of all intracellular protein degradation by the proteasome. This critical role in maintaining protein homeostasis is emphasized by links between CRLs and diseases such as cancer and neurodegenerative disorders, inflammation, host-pathogen interactions, and the immune response. The activation of CRLs by covalent modification with the ubiquitin-like protein NEDD8 functions as a major regulatory mechanism to control these enzymes. NEDD8 modifications are essential for cancer cell survival as a small molecule known as MLN4924 that generally inhibits all neddylation pathways selectively induces cancer cell death by triggering S-phase DNA re-replication and inhibits tumor growth in xenograft studies.
UBC12, a member of the 50+ ubiquitin and ubiquitin-like protein conjugating enzyme (E2) family, is responsible for the direct covalent modification of a subset of cullins with NEDD8 and represents a novel target for small molecule probes. Although E2s have been considered unfavorable targets for probe development due to the absence of obvious catalytic pockets or small molecule binding sites, recent work on the ubiquitin E2 Cdc34 identified a cryptic binding pocket that when occupied by the small molecule CC0651 inhibits the covalent transfer of ubiquitin by preventing a variety of subtle allosteric changes throughout the enzyme. Intriguingly, similar pockets can now be discerned in other E2 structures including UBC12. However, the critical residues necessary for CC0651 binding to Cdc34 are not conserved and it remains unknown if this allosteric activation is a general mechanism used by all E2s or is specific to Cdc34.
The overall objective of this proposal is to identify small molecule probes that inhibit both ATP dependent Nedd8 activation of NAE (E1) and UBC12(E2)-mediated transfer of NEDD8 from UBC12 onto its cullin substrates. This is done in a TR-FRET assay.
1. Watson IR, Irwin MS, Ohh M. NEDD8 pathways in cancer, Sine Quibus Non. Cancer Cell. 2011;19(2):168- 76.
2. Schulman BA, Harper JW. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. Nat Rev Mol Cell Biol. 2009;10(5):319-31. PMCID: PMC2712597.
3. Pan ZQ, Kentsis A, Dias DC, Yamoah K, Wu K. Nedd8 on cullin: building an expressway to protein destruction. Oncogene. 2004;23(11):1985-97.
4. Petroski MD, Deshaies RJ. Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol. 2005;6(1):9-20.
5. Duda DM, Borg LA, Scott DC, Hunt HW, Hammel M, Schulman BA. Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation. Cell. 2008;134(6):995-1006. PMCID: 2628631.
6. Furukawa M, Zhang Y, McCarville J, Ohta T, Xiong Y. The CUL1 C-terminal sequence and ROC1 are required for efficient nuclear accumulation, NEDD8 modification, and ubiquitin ligase activity of CUL1. Mol Cell Biol. 2000;20(21):8185-97. PMCID: 86428.
Baculovirus expressed proteins from insect cells: NAE and CRL1SKP2 , FLAG-tagged-CUL1 and the SKP1/SKP2 receptor complex; Bacterially expressed: UBC12 and NEDD8; ATP Terbium (Tb) anti-FLAG antibody (Cisbio) & fluorescein NEDD8 (fNEDD8, Boston Biochem)
This "NAE/UBC12 Assay" includes the pre-activation step for the ATP dependent preactivation of NAE for Nedd8 charging, so will interrogate both inhibitors of NAE (E1)activation and UBC12 (E2) Neddylation activity
Reaction buffer: 25 mM HEPES pH 7.8, 50 mM NaCl, 1 mM TCEP, 1 mM MgCl2, with 0.005% Tween 20.
Procedure: Tb anti-FLAG antibody pre-incubated 1 hr with CRL1SKP2 at 25 masculineC to allow binding, followed by addition of ALL other components into a 384-well plate. The TR-FRET signal reflecting the UBC12-catalyzed trans-thiolation reaction was measured on a BMG Labtech PHERAstar (Lanthascreen optical module, ex337/em520/em490).
Compounds with either:
1. %Activity_Corrected >=40% and 0.1 < FRatio < 1.5
2. Z score <-5 or 0.1
Are considered to be active in this assay.
The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
** Test Concentration.
Data Table (Concise)